The expression of a gene in both prokaryotic and eukaryotic organisms involves first the synthesis of RNA from a DNA template followed by protein synthesis from the RNA.
Transcription, the synthesis of RNA from a DNA template and the first step in the expression of a gene, is controlled by certain signals present on the DNA. These signals are nucleotide sequences which initiate transcription and control the amount of transcription taking place at a given time. The control signals generally consist of promoter and operator regions. The promoter region is a site that is specific for the binding of RNA polymerase and is the initiation point for transcription. Operators function in conjunction with a repressor to control the amount of transcription.
Transcription of a DNA segment is effected by the enzyme RNA polymerase. After RNA polymerase binds to the promoter at the -35 and -10 recognition regions (M. Rosenberg and D. Court, Ann. Rev. Genet. 13:319-353, 1979), it transcribes nucleotides which encode a ribosome binding site and translation initiation signal and then transcribes the nucleotides which encode the actual structural gene until it reaches so-called stop signals at the end of the structural gene. The RNA polymerase acts by moving along the DNA segment and synthesizing single-stranded messenger RNA (mRNA) complementary to the DNA. As the mRNA is produced, it is bound by ribosomes at the ribosome binding site (also called the Shine-Dalgarno region). The ribosomes translate the mRNA, beginning at the translation initiation signal and ending at the stop signals, to produce a polypeptide having the amino acid sequence encoded by the DNA.
Through the use of genetic engineering techniques genes from one organism can be removed from that organism and spliced into the genetic information of a second organism and the polypeptide encoded by that gene expressed by the second organism. It is desirous to maximize the expression of the foreign gene and thus obtain high yields of the resultant polypeptide. It has been realized that one way in which gene expression can be regulated is through selection and manipulation of the control signals discussed above.
There is variation among different promoters in their strength and their ability to be repressed efficiently. A promoter which cannot be repressed easily is of only limited use with genes whose protein product in small amounts is toxic to the cell or inhibits maintenance of the plasmid. In such situations, maximal repression of the genes is needed to assure that the host cell and/or plasmid can grow normally until derepression is desired.
Some promoters also suffer a disadvantage when they are present on multi-copy plasmids in that they cannot be repressed efficiently unless a suitable repressor also is located on that plasmid and thus present in multiple copies.
Such promoters are in contrast to others which can be repressed fully by the amount of repressor made from a single chromosomal gene copy. These promoters, however, may have other drawbacks. They may not, for example, be as strong as other promoters.
Various efforts have been made to manipulate different promoter/operator systems so as to enhance promoter strength or increase efficiency of repression. European Patent Application 067,540 (see also De Boer et al. in "Promoters: Structure and Function," ed. R.L. Rodriguez, M.J. Chamberlin, Praeger, 1982, pp. 462-481), for example, describes and claims a hybrid promoter/operator. This hybrid is constructed by ligating the -10 region of one promoter/operator sequence, capable of being derepressed by induction, downstream from a DNA fragment which comprises the -35 region and 5' flanking region of a second promoter which has a stronger signal sequence than the first promoter/operator sequence. The two DNA fragments are linked at a position between about the -35 and -10 recognition sequences for binding of RNA polymerase to the promoter/operator sequence. The fusion results in an entirely new promoter sequence.
Although such a hybrid promoter/operator can be used advantageously in certain situations, it still may prove to be unsatisfactory in others. For example, although the transcription efficiency of the promoter contributing the -10 region may be enhanced, the promoter may not be regulated as tightly as desired under certain circumstances.
There thus remains a need for a regulatory sequence that has a strong promoter which can be repressed highly efficiently. Accordingly, it is an object of this invention to construct a novel regulatory region having these characteristics. It also is an object of this invention to construct such a regulatory region that can be ligated conveniently to a variety of prokaryotic and eukaryotic genes.